Gramicidin A is hydrolyzed by a d‐stereospecific peptidase produced by Bacillus anthracis

Summary Previously we described the discovery of a Bacillus spp. specific peptidase activity related to d‐stereospecific peptidases (DSPs). The peptidase showed a strong preference for d‐leucine and d‐valine amino acids. These amino acids are present in the structure of the non‐ribosomal peptide (NRP) antibiotics gramicidin A, B and C and polymyxin E. To examine if the Bacillus spp. DSP‐related peptidase can hydrolyze these NRPs, the effect of gramicidin A and C and polymyxin E on peptidase activity in Bacillus anthracis culture supernatant was monitored. It was found that both gramicidins inhibited the DSP‐related activity in a competitive manner. MALDI‐TOF analysis revealed that upon incubation with B. anthracis culture supernatant gramicidin A hydrolyzation products appeared. This study shows that the Bacillus spp. specific DSP‐like peptidase was potentially produced by the bacteria to gain intrinsic resistance against NRP antibiotics. These results are of utmost importance in research towards antimicrobial resistance, whereas transfer of DSP‐related activity to other clinically relevant pathogens can be a serious threat to human health.

Previously we described the discovery of a Bacillus spp. specific peptidase activity related to Dstereospecific peptidases (DSPs). The peptidase showed a strong preference for D-leucine and D-valine amino acids. These amino acids are present in the structure of the non-ribosomal peptide (NRP) antibiotics gramicidin A, B and C and polymyxin E. To examine if the Bacillus spp. DSP-related peptidase can hydrolyze these NRPs, the effect of gramicidin A and C and polymyxin E on peptidase activity in Bacillus anthracis culture supernatant was monitored. It was found that both gramicidins inhibited the DSPrelated activity in a competitive manner. MALDI-TOF analysis revealed that upon incubation with B. anthracis culture supernatant gramicidin A hydrolyzation products appeared. This study shows that the Bacillus spp. specific DSP-like peptidase was potentially produced by the bacteria to gain intrinsic resistance against NRP antibiotics. These results are of utmost importance in research towards antimicrobial resistance, whereas transfer of DSP-related activity to other clinically relevant pathogens can be a serious threat to human health.

Background
Soil contains a large scale of microorganisms among fungi, amoebae and bacteria, such as Bacillus spp. and Actinomyces spp. There, these microorganisms compete for limited resources such as nutrients and water. To eliminate their competitors several soil micro-organisms produce antibiotics. Especially Actinomycetes spp. are well known for their intensive antibiotic production in soil. For instance, streptomycin, an aminoglycoside, was isolated from the actinomycete and soil-inhabiting filamentous bacterium Streptomyces griseus (Schatz et al., 2005). Another source of natural occurring antibiotics comprises the class of non-ribosomal peptides (NRPs) produced by Bacillus spp. (Sumi et al., 2015), such as polymyxin B and E (colistin), bacitracin, gramicidin A-C and tyrocidine A-C (Stansly and Schlosser, 1947;Toscano and Storm, 1982). These NRPs contain D-amino acids in their structure to ensure proteolytic stability and to expand their bioactive spectrum (Nagata, 1999;Schwarzer et al., 2003).
Earlier we described the discovery of Bacillus spp. peptidase activity related to the degradation of D-amino acids containing fluorogenic peptide substrates (Kaman et al., 2011). Based on the abundant presence of Damino acids in the peptidoglycan layer of the bacterial cell wall we were first tempted to speculate that this peptidase might play a role in bacterial cell wall synthesis and remodelling. However, recent in-depth literature search disclosed the fact that virtually all NRP antibiotics produced by Bacillus spp. contain D-amino acids (Nagata, 1999). This led to the renewed hypothesis that the previously found Bacillus spp. D-stereospecific peptidase (DSP) related activity might be produced by the bacteria to hydrolyze NRP antibiotics in order to gain intrinsic resistance.

Results and discussion
Previously, we described the discovery of DSP activity by Bacillus spp. (Kaman et al., 2011). Pinpointing substrate specificity using a fluorogenic peptide substrate library disclosed a preference for D-leucine and D-valine containing substrates (Kaman et al., 2013). Peptidase activity was detected in culture supernatants of Bacillus anthracis, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus licheniformis. In contrast, no degradation of D-leucine/Dvaline containing substrates was found in culture supernatants of Bacillus atrophaeus (former Bacillus globigii) and Bacillus subtilis (Kaman et al., 2011).
To further investigate these findings the hydrolysis of seven D-leucine and D-valine containing fluorogenic substrates (Kaman et al., 2013) was analysed using culture supernatant of B. anthracis. It was found that the two Dleucine containing substrates, with an N-terminal Lglycine or L-leucine, were degraded with comparable efficiency (Fig. 1A). Of the five D-valine containing substrates the substrate with an N-terminal L-valine was degraded with the highest efficiency (Fig. 1A). The Dvaline containing substrates with an N-terminal L-serine or glycine were not hydrolyzed (F/min < 5) by B. anthracis culture supernatant (Fig. 1A). The culture medium itself, BHI, was found to be devoid of d-leucine/ d-valine proteolytic activity (data not shown). These results were comparable to our earlier findings (Kaman et al., 2011;Kaman et al., 2013). Furthermore, to characterize the peptidase, we examined the effects of several (specific) protease inhibitors on the D-leucine/Dvaline proteolytic activity. Upon addition of EDTA, a metalloprotease inhibitor, a two-fold increase in peptidase activity was observed (Fig. 1B). This indicated that the activity of the peptidase is in a negative manner influenced by metal ions. The addition of the cysteine protease inhibitor NEM slightly affected peptidase activity. In contrast, the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) completely inhibited D-leucine/D-valine proteolytic activity. These results suggest that the D-leucine/D-valine peptidase belongs to the family of serine proteases and that its activity is potentially regulated by metal ions. Regulation of peptidase activity by metal ions is a known mechanism. For example, the activity of the serine protease HtrA produced by the bacterium Helicobacter pylori is inhibited by zinc and copper ions (Bernegger et al., 2020). Also, Higaki et al. (1990) observed regulation of the activity of a serine protease by a metal switch.
To gain more insight into the functionality of the D-leucine/ D-valine proteolytic activity, protease production was monitored during growth. B. anthracis and B. subtilis showed similar growth curves with a decrease in OD600 at 48 h ( Fig. 1C). B. anthracis showed proteolytic activity which correlated with the increase in OD600. No degradation of the substrates was detected using the B. subtilis culture supernatant ( Fig. 1D).  Bacillus anthracis produces a D-leucine/D-valine recognizing serine peptidase. Bacillus anthracis culture supernatant was incubated with all D-leucine/D-valine containing substrates present in the FRET-peptide library. Proteolytic activity was defined in F min À1 (A). The effect of the protease inhibitors EDTA (5 mM), NEM (10 mM) and PMSF (10 mM) on the hydrolysis of the three D-leucine/D-valine substrates degraded with the highest efficiency. Substrate hydrolyzation without the addition of inhibitor was taken as the 'normalized' (100%) value (B). Proteolytic activity during growth of B. anthracis (black) and B. subtilis (grey) was monitored. Bacteria were grown for 48 h in BHI at 35 C and samples were taken to analyse growth (C) and proteolytic activity (D). Proteolytic activity was measured using the substrates Gly-D-Leu and Val-D-Val. Results are expressed as mean AE SEM (n = 3).
We postulate that the D-leucine/D-valine proteolytic activity detected in culture supernatants of Bacillus spp. might lead, or contribute to, degradation and inactivation of the D-valine/D-leucine containing antibiotics gramicidin and polymyxin E, which contain both one or more Dvaline and/or D-leucine residues in their structure (Fig. 2). This would be in line with our findings that B. anthracis is resistant against gramicidin A (MIC >10 μM, data not shown). In the same experiment the susceptibility of B. subtilis towards gramicidin A was determined. In contrast to the results of Wang et al. (2012) we found that the B. subtilis strain used in this study also had a MIC of >10 μM (data not shown). However, the secretion of a Dleucine/D-valine peptidase by B. subtilis to degrade Dvaline/D-leucine containing antibiotics is unlikely, whereas this bacterium produces the D-leucine containing antibiotic surfactin (Tojo et al., 2015). This suggests that B. subtilis and B. anthracis use different mechanisms to gain resistance against gramicidin A.
To check the aforementioned hypothesis the effect of gramicidin A, C and polymyxin E on the activity of the Dleucine/D-valine peptidase was examined. Gramicidin B, however, was not included, whereas this antibiotic was not commercially available. It was found that both gramicidin A and C inhibited the D-leucine/D-valine proteolytic activity on all tested substrates (Fig. 3A-C). In contrast, no inhibition of proteolytic activity was observed when polymyxin E was added to the peptidase assay. This might be related to the fact that in the structure of polymyxin E N-terminal from D-leucine the unnatural amino acid L-α,γ-diaminobutyric acid (L-Dab) is present (Fig. 2). L-Dab is not commonly recognized by peptidases and thereby potentially protects polymyxin from hydrolyzation by the D-leucine/D-valine peptidase (Lu et al., 2020). The type of inhibition by gramicidin A was studied using two fluorogenic peptide substrates in which a comparable amino acid motif was present as in gramicidin A; valine-D-valine and glycine-D-leucine (i.e. glycine-isoleucine in gramicidin A) (Fig. 2). Analysing the enzyme kinetics the Lineweaver-Burk plot showed that gramicidin A inhibits D-leucine/D-valine proteolytic activity in a competitive manner ( Fig. 3D and E). This indicates that gramicidin A competes with the D-leucine/D-valine substrates for binding to the Bacillus spp. peptidase and suggests that gramicidin A might be the natural substrate for the D-leucine/Dvaline peptidase produced by B. anthracis. To further investigate this hypothesis gramicidin A was incubated with culture supernatant of B. anthracis. As negative control culture supernatant from B. subtilis, a Bacillus spp. which lacks D-leucine/D-valine peptidase production, was used (Kaman et al., 2011). MALDI-TOF analysis revealed that gramicidin A (m/z 1905 X = L-Val/m/z 1921 X = L-Ile) was indeed degraded by B. anthracis culture supernatant (Fig. 4A). No degradation products were observed when gramicidin A was incubated with culture supernatant of B. subtilis (Fig. 4B). The sizes of the degradation products in the gramicidin A sample incubated with B. anthracis supernatant were m/z 1726 (P1/X = L-Val), m/z 1743 (P2/X = L-Ile) and m/z 1501 (P3/X = unknown). The peak sizes of P1/P2 correspond with C-terminal hydrolyzation of gramicidin A between D-leucine (L14) and L-tryptophan (W15). To confirm this cleavage position and to unravel the identity of peak P3 additional experiments are needed. Whereas no peaks were observed in the Bacillus supernatant alone samples, we can conclude that the peaks present in the gramicidin A/B. anthracis sample originate from gramicidin A (Fig. 4A  and B).
That Bacillus spp. are able to protect themselves against the antimicrobial effects of NRP antibiotics produced by hydrolyzation has already been described by others (Li et al., 2018;Yin et al., 2019). The hydrolyzation mechanism discovered in the study of Li et al. is in line with the results of our study; using a networkingassociated genome-mining platform Li et al. found two Dstereoselective peptidases produced by bacteria of the order of Bacillales; TriF (Paenibacillus polymyxa) and BogQ (Brevibacillus laterosporus). Additionally, like BogQ, the D-leucine/D-valine peptidase described in this study belongs to the family of serine peptidases. According to the MEROPS database, the genome of B. anthracis contains 15 known or putative serine peptidases which are absent in the genome of B. subtilis (Table S1). Six of these B. anthracis-specific serine peptidases belong to the subfamily S12, of which BogQ is a member. Potentially the degradation of the D-leucine/Dvaline substrates is related to the activity of one of these six S12 peptidases but additional research is needed to unravel the identity of the DSP peptidase described in this study.
Our results show that a range of Bacillus spp., among which the biowarfare associated pathogen B. anthracis, produce a peptidase with DSP related activity. That also other bacteria in the order of Bacillales can produce NRP hydrolyzing DSPs was to be expected; 403 putative peptidases with similar domain compositions to BogQ are present in the order of Bacillales (Li et al., 2018). The DSP-related activity we discovered in 2011 differs from BogQ and TriF in D-amino acid preference. Where TriF hydrolyzes the C-terminal side of aromatic D-amino acids  and BogQ cleaves peptides at the C-terminal side of cationic D-amino acids the DSP from B. anthracis shows a preference for D-leucine and D-valine (both uncharged and hydrophobic) residues. However, the exact cleavage preferences of the B. anthracis DSP, its identity and if the observed hydrolyzation of gramicidin A leads to inactivation of the antibiotic remain to be elucidated. The observation that different types of DSPs, with a variety in recognition sequences, are produced by different bacteria of the Bacillales order is of utmost importance in research towards antimicrobial resistance. DSPs can potentially be transferred to other, clinically relevant, pathogens and thereby be a serious threat to human health. Additionally, the existence of these DSPS should be taken into account when new antibiotics are designed and for example D-amino acids are added to their structure to improve stability (Molhoek et al., 2011;Bann et al., 2019).

Bacteria
Bacillus anthracis Vollum (NCTC 10340) and Bacillus subtilis (ATCC 6051) were grown overnight in 15 ml Brain Heart Infusion (BHI) medium (BioTrading, Mijdrecht, The Netherlands) at 35 C (200 rpm). For the growth curve experiment bacteria were grown in 10 ml BHI for 48 h at 35 C (200 rpm). Samples were taken at 0, 2, 4, 6, 24 and 48 h and absorbance was measured at 600 nm using a spectrophotometer (Ultrospec10, Amersham Biosciences, UK). Next, the bacteria were pelleted by centrifugation for 20 min at 3250g. Supernatant was filtered through a 0.22 μM filter (Millipore, Amsterdam, The Netherlands) and sterility was verified by plating 1/10 of the stock on TSA plates and subsequently incubation for 7 days at 35 C. Culture supernatant was stored at À20 C until sterility was confirmed.

Peptidase assay
Proteolytic activity was determined, as described earlier (Kaman et al., 2011). Fluorescence was read for 60 min at 2 min intervals using a fluorescence microplate reader (FLUOstar Galaxy, BMG Laboratories, Offenburg, Germany) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Peptidase activity was defined in fluorescence per minute (F min À1 ). BHI medium was used as negative control. Characterization of the peptidase was performed by the addition of specific protease inhibitors to the assay; 5 mM EDTA, 10 mM N-ethylmaleimide (NEM) or 10 mM PMSF. None of the protease inhibitors influenced the pH of the reaction mixture. To determine the effect of D-leucine/D-valine containing antibiotics on proteolytic activity, B. anthracis culture supernatant was incubated with 16 μM FRETpeptide substrate in the presence of varying concentrations of polymyxin E, gramicidin A and gramicidin C from Brevibacillus brevis (all purchased from Merck Life Science, Amsterdam, The Netherlands). Peptidase activity was defined as a percentage of the control (proteolytic activity without antibiotics). To determine the type of inhibition (competitive or non-competitive) 0-200 μg ml À1 gramicidin A was incubated with 1-16 μM FRET-peptide substrate. The initial velocity was plotted against the substrate concentration using the Michealis-Menten equation and GraphPad Prism8 software. All measurements were performed in triplicate, and data are presented as mean AE SEM.

MALDI-TOF analysis
Culture supernatants of B. anthracis and B. subtilis were incubated with 1 mg ml À1 gramicidin A for 72 h at 37 C. Culture supernatant without gramicidin A and 50 mg ml À1 gramicidin A in water were used as controls. Following incubation the samples were purified using Pierce C18 ZipTips (Thermo Fisher Scientific, Breda, The Netherlands) according to the manufacturer's instructions. Briefly, ZipTips were preconditioned with 10 μl 100% acetonitrile and subsequently washed three times with 10 μl 10% (vol./vol.) acetonitrile in water. After the application of 10 μl sample the ZipTips were again washed three times with 10 μl 10% (vol./vol.) acetonitrile in water followed by elution with 10 μl 80% (vol./vol.) acetonitrile in water. The purified samples were analysed using MALDI-TOF (Bruker Daltonik GmbH, Bremen, Germany).